A regulatory protein functionally associated with DNA polymerase alpha has recently been isolated from calf thymus. In the presence of this protein, deoxyribonucleoside triphosphates are effective inhibitors of DNA polymerase in addition to their substrate function. DNA polymerase-regulatory protein activity, measured by using polydeoxy-adenylate-oligodeoxythymidylate as template-primer, is inhibited by dGTP in a parabolic-competitive fashion (Ki equals 15 micronmoles) and by dCTP in a linear-competitive manner (Ki equals 162 micromoles). Neither the four natural ribonucleoside triphosphates nor dATP inhibited the DNA polymerase-regulatory protein system to any significant extent. These results indicate that deoxyribonucleoside triphosphate pools may be involved in the regulation of cellular DNA synthesis through a direct effect on DNA polymerization. The further purification and characterization of this protein is proposed. An appropriate kinetic equation for DNA polymerase will be developed following kinetic analysis of the enzyme using homopolymers as template. The effects of various anti-metabolites, especially arabinosylcytosine, on the DNA polymerase-regulatory protein system will be evaluated. These studies are designed to determine the importance and function of this protein with respect to the relationship between deoxyribonucleoside triphosphate pools and DNA synthesis, the mechanism of action of antimetabolites and a potentially critical role in DNA replication.